Tandem Mass Tag-Based Phosphoproteomics in Plants

Mass spectrometry-based proteomics provide a powerful tool for plant research, allowing global detection of steady-state levels of proteins under a given experimental setup. Here, we provide an optimized protocol for proteomic profiling using tandem mass tag (TMT) labeling followed by liquid chromatography-mass spectrometry (LC-MS/MS) to quantitate phosphopeptides and non-phosphopeptides from the same samples. The outlined protocol comprises a series of successive steps, namely, SDS (sodium dodecyl sulfate) protein extraction, protein precipitation, digestion, TMT labeling, phosphopeptide enrichment, high pH reversed-phase fractionation, LC-MS/MS analysis, protein identification, and data analysis. Our proteome-scale protocol requires 0.1 mg protein per sample and allows for the reliable and accurate quantification of more than 8000 proteins in Arabidopsis plant samples across multiple conditions, including low abundant peptides.